This invention relates to methods for detecting type II collagen fragments in biological media. More specifically, it relates to methods for quantifying protein fragments found in urine resulting from cleavage of type II collagen.
The physiological turnover of articular cartilage represents a fine balance between synthesis and degradation. It is a feature of normal growth and development and maintenance of cartilage in the adult. Net cartilage loss is a feature of arthritis. It is strongly associated with disability and a low quality of life. Cartilage destruction in rheumatoid arthritis and osteoarthritis is currently diagnosed based on combined clinical symptoms and radiological findings. Damage to articular cartilage occurs early in the disease, long before it can be detected radiologically; damage is detected radiologically only after there is extensive and probably irreversible cartilage loss. Therefore, it is of critical importance that clinicians have biochemical markers for early diagnosis of cartilage damage so therapy can be initiated early, before extensive damage is done. Furthermore, such markers can be used in the design of clinical trials in the selection of subjects for enrollment who have a higher likelihood of rapid progression. In addition, treatment effects could be monitored in a timely manner. TIINE analysis may also provide information by which compound effectiveness may be measured.
Type II collagen constitutes the bulk of the fibrillar backbone of the cartilage matrix, just as type I collagen forms the fibrillar organization of the extracellular matrix of most other tissues such as skin, bone, ligaments and tendons.The destruction of articular cartilage during arthritic disease is due, in part, to the degradation of the extracellular matrix, which is composed primarily of fibrillar type II collagen and aggregating proteoglycans. In articular cartilage, type II collagen fibrils are responsible for the tensile strength and structure whereas the proteoglycans provide the compressive stiffness necessary for normal articulation and function. The precise mechanisms by which these connective tissue components are degraded are not fully understood. In mammals, an important mechanism involves the collagenases, MMP-1, MMP-8, MMP-13 and MT1-MMP, which are a group of enzymes capable of site-specific cleavage of helical (native) collagen. All three type of collagen are composed of a tightly wound triple helix, which in man, are cleaved extracellularly by the collagenases to produce xc2xe and xc2xc length cc-chain fragments that are identifiable by polyacrylamide gel electrophoresis.
In a first aspect, the present invention provides a method for monitoring urine for type II collagen fragments, said method comprising contacting said urine with a capture antibody, wherein said capture antibody binds specifically to type II collagen fragments, to the substantial exclusion of any binding to type I or III collagen fragments, contacting said urine with a detection antibody, wherein said detection antibody binds specifically to collagenase-generated collagen fragments, and detecting the amount of type II collagen fragments bound to said capture and detection antibodies.
In a preferred embodiment of the first aspect, the detection antibody is active against the sequences set forth in SEQ ID NOS: 1 and 2.
In another preferred embodiment of the first aspect, the detection antibody has a VH sequence at least 95% homologous to that set forth in SEQ ID NO: 32, and a VL sequence at least 95% homologous to that set forth in SEQ ID NO: 33.
In another preferred embodiment of the first aspect, the detection antibody has the same CDRs as the VH sequence set forth in SEQ ID NO: 32, and the same CDRs as the VL sequence set forth in SEQ ID NO: 33.
In another preferred embodiment of the first aspect, the capture antibody is active against the sequences set forth in SEQ ID NOS: 3 and 4.
In another preferred embodiment of the first aspect, the capture antibody has a VH sequence at least 95% homologous to that set forth in SEQ ID NO: 48, and a VL sequence at least 95% homologous to that set forth in SEQ ID NO: 49.
In another preferred embodiment of the first aspect, the capture antibody has the same CDRs as the VH sequence set forth in SEQ ID NO: 48, and the same CDRs as the VL sequence set forth in SEQ ID NO: 49.
Those skilled in this art will recognize that the order of contacting the antibodies with the biological media may be reversed.
Furthermore, in certain embodiments, this aspect of the invention comprises a method for monitoring urine for type II collagen fragments as described above, wherein said contacting steps occur simultaneously.
In another preferred embodiment of the first aspect, the antibody contacting steps occur sequentially, and after the contacting step involving the capture antibody, and prior to the contacting step involving the detection antibody, said capture antibody is immobilized onto a magnetic material.
In another preferred embodiment of the first aspect, the method as described above includes the further steps of contacting a series of control samples with said capture antibody, contacting said control samples with said detection antibody, and detecting the amount of type II collagen fragments in said control samples bound to said capture and detection antibodies.
In a second aspect of the present invention, there is provided a kit for monitoring urine for type II collagen fragments, said kit comprising a capture antibody, wherein said capture antibody binds specifically to type II collagen fragments, to the substantial exclusion of any binding to type I or III collagen fragments, a detection antibody, wherein said detection antibody binds specifically to collagenase-generated collagen fragments, a container, and instructions describing a method of using said first antibody and said second antibody to monitor biological media for type II collagen fragments.
In a preferred embodiment of the second aspect, said detection antibody has a VH sequence at least 95% homologous to that set forth in SEQ ID NO: 32, and a VL sequence at least 95% homologous to that set forth in SEQ ID NO: 33, and said capture antibody has a VH sequence at least 95% homologous to that set forth in SEQ ID NO: 48, and a VL sequence at least 95% homologous to that set forth in SEQ ID NO: 49.
In another preferred embodiment of the second aspect, said detection antibody has the same CDRs as the VH sequence set forth in SEQ ID NO: 32, and the same CDRs as the VL sequence set forth in SEQ ID NO: 33, and said capture antibody has the same CDRs as the VH sequence set forth in SEQ ID NO: 48, and the same CDRs as the VL sequence set forth in SEQ ID NO: 49.
In another preferred embodiment of the second aspect, said kit further comprises a positive control fluid comprising control urine containing a known amount of type II collagen fragments, and a dilution fluid comprising control urine for diluting samples being tested with said kit.
In a third aspect of the present invention there is provided a biosensor chip for detecting the presence of an immunological binding event, wherein said chip comprises a first antibody active against the sequences set forth in SEQ ID NOS: 1 or 2, or a second antibody active against the sequences set forth in SEQ ID NOS: 3 or 4.
In a fourth aspect of the present invention there is provided a method of diagnosing a patient for a disease state associated with the degradation of type II collagen, said method comprising the step of detecting the presence of atypically large amounts of type II collagen fragments in urine collected from said patient.
In a fifth aspect of the present invention there is provided a method of performing a clinical trial to evaluate a drug believed to be useful in treating a disease state associated with the degradation of type II collagen, said method comprising measuring the level of type II collagen fragments in urine collected from a set of patients, administering said drug to a first subset of said patients, and a placebo to a second subset of said patients, repeating said measuring step after the administration of said drug or said placebo, and determining if said drug is reducing the amount of type II collagen fragments present in said urine of said first subset of patients to a degree that is statistically significant as compared to any reduction occurring in said second subset of patients, wherein a statistically significant reduction indicates that said drug is useful in treating said disease state.
Useful in the present invention is a hybridoma cell line or E. coli culture that produces a monoclonal antibody that binds to peptides consisting essentially of the structure as set forth in the Sequence Listing as SEQ ID NO: 1 or SEQ ID NO: 2, the cell line having the identifying characteristics of ATCC HB-12436.
Also useful in the present invention is a hybridoma cell line or E. coli culture that produces a monoclonal antibody that binds to peptides consisting essentially of the structure as set forth in the Sequence Listing as SEQ ID NOS: 3 or 4, the cell line having the identifying characteristics of ATCC HB-12435.